The 174-base bacteriophage ?29 prohead RNA (pRNA) is an essential component of the motor that packages the 19-kilobase pair genomic DNA-gp3 complex (DNA-gp3) into the viral precursor capsid (prohead). This motor is one of the strongest molecular motors known, generating ~110 pN of force. pRNA forms a novel cyclic hexamer by intermolecular base pairing of identical molecules. This multimer binds to the head-tail connector of the prohead, where it appears as a pentameric ring by cryoEM-3D reconstruction. A multimer of the ATPase gp16 then binds to the pRNA oligomer to constitute the ATP-hydrolyzing subunits of the motor. pRNA is hypothesized to function in docking of the DNA-gp3 on the prohead, in recognition of the left end of DNA-gp3 to initiate packaging, and in assembly and enzyme function of the DNA translocating ATPase. pRNA exits the DNA-filled head during neck and tail assembly, and it is not a part of the mature virion. Study of the structure and function of this RNA-dependent DNA packaging motor may have general significance in uncovering targets for antiviral agents. The ultimate goal of the research is to determine the structural and functional roles of pRNA in the mechanism of DNA translocation. The current aims are: 1) X-ray crystallography will be used to determine atomic structures of monomeric and oligomeric pRNAs;2) NMR will be used to determine the structure of the pRNA intermolecular pseudoknot and the essential CCA bulge of the A-helix;3) Solution and site-directed hydroxyl radical probing will be used to map pRNA nucleotides in prohead/pRNA complexes with and without the ATPase gp16;4) Site-directed and random pRNA mutants will be isolated to interrogate function of the pRNA in DNA translocation;and 5) Single-molecule laser tweezers studies of pRNA mutants having partial function will probe the roles of pRNA in ?29 DNA translocation. PUBLIC HEALTH RELEVANCE: There is a paucity of antiviral agents, and the design of new drugs depends upon a thorough understanding of the basic mechanisms of viral infection and assembly. The infection and assembly mechanisms of the bacteriophage ?29 serve as models for understanding the infection and assembly mechanisms of dsDNA viruses such as herpesvirus and adenovirus, which have significant commonalities to ?29. The assembly processes of ?29 studied here, such as DNA packaging, are targets for antiviral agents.